Fluorescence detection offers one of the most sensitive methods for quantification of probe molecules in biological and material systems because it can attain near single-molecule sensitivity levels. Consequently the technique is widely used in the assaying of biochemical and cellular systems, and in particular the microscopic imaging of cell-based assay, where rich biological information is provided from multiplexed high-content data (Ramm P, “Image-based screening: a technology in transition,” Curr. Opin. Biotech. 16, 41-48 (2005); Zhou X and Wong STC, “High content cellular imaging for drug development,” IEEE Sig. Proc. Mag. 23, 170-174 (2006)).
Attainment of high sensitivity levels requires rejection of background light. In part, background arises from the scatter of the excitation light. Better filtering and separation of excitation and emission lights reduces background, but a small amount can still leak through to the detector. Background also arises from spurious fluorescence, from components of the sample other than the probe of interest, from the sample holder, and from the measuring instrument's optical components. Because of its composite nature, spurious fluorescence generally occurs over a broad range of wavelengths and its removal by spectral filtering is not very effective.
One way of reducing background is by time resolution of the signal, which amounts to temporal filtering of the signal. When the fluorescent probe is appropriately long-lived and the excitation light is pulsed (or modulated with high frequency), test sample fluorescence will last longer than the scattered excitation, or spurious fluorescence. The effect is particularly pronounced with time-resolved fluorescence (TRF) reagents, where by design of chemistry, sample probes have fluorescence lifetimes in the ms (10−3 s) to μs (10−6 s) time domain (Hemmila I, Laitala V, “Progress in lanthanides as luminescent probes,” J Fluores 15, 529-542 (2005); Hemmila I, Mukkala V-M, “Time-resolution in fluorometry technologies, labels and applications in bioanalytical assays,” Crit Rev Clin Lab Sci 38, 441-519 (2001)). In comparison, background from the scattered excitation pulse and spurious fluorescence lasts for a shorter period of time, approximately for the duration of the excitation pulse itself. A key requirement for time resolution of fluorescence is that the duration of the excitation pulse should be less than that of the test compound. Time resolution leads to very significant improvements in sensitivity. In practice, for example, one finds that TRF imagers such as the LEADSEEKER™ manufactured by GE® Healthcare located in Piscataway, N.J. have detection sensitivities some two orders of magnitude higher than the same system in its steady-state fluorescence detection mode.
Apart from the rejection of background light, there are additional advantages to time resolved measurements. Fluorescence is usually measured (or imaged) as steady-state fluorescence (SSF). That is, a steady source of excitation light is used to generate a constant flux of sample fluorescence. The SSF method suffers because of two reasons: (a) The signal depends on the intensity of the excitation source, and the design details of the optical measuring instrument. That is, SSF signal values are dependent on the particulars of the measuring system and hence not reproducible across different laboratories. In contrast, time resolved measurements can yield the mean fluorescence lifetime of the probe, a molecular property which has values independent of the measuring system, and hence reproducible across different laboratories.
Moreover, SSF only gives information on the average state of an excited probe over long time periods. Much more information about the dynamics of a probe and its microenvironment may be obtained if its fluorescence is followed by time-resolution. Examples of dynamics include kinetics of molecular rotation, diffusion, reaction, energy transfer, etc. Of special interest is the rotational depolarization behavior of long-lived TRF reagents. This is because in the usual fluorescence polarization (FP) assays one relies on ns lifetime probes. In this time regime one can only interrogate the rotational dynamics of small-molecules. As a result such FP assays can not detect changes to the structure of a large protein, because the rotational time scales would be too long to affect the polarization of fluorescence. Long-lived TRF reagents however have lifetimes about 105 times longer than ns dyes and can widen the applicability of FP assays, particularly within cell-based systems (Owicki J C, “Fluorescence polarization and anisotropy in high throughput screening: perspective and primer,” J Biomol Scr 5, 297-306 (2000); Austin R H, Chan S S, Jovin T M, “Rotational diffusion of cell surface components by time-resolved phosphorescence anisotropy,” Proc Natl Acad Sci. 76, 5650-5654 (1979)).
Yet another application of time-resolved detection is for label-free detection of proteins in biochemical and cellular media. Here one relies on the intrinsic long-lived phosphorescence of cellular proteins (ms and longer), particularly from the tryptophan residues (Vanderkooi J M, Tryptophan phosphorescence from proteins at room temperature, In “Topics in Fluorescence Spectroscopy,” Volume 3 Biochemical Applications, Lakowicz J R Edited, Plenum Press, NY, 1992, pp. 113-136; Vanderkooi J M et al., “On the prevalence of room temperature protein phosphorescence,” Science 236, 568-569 (1987)). Lack of sensitive time-resolved imaging systems has hampered the development of novel assays for the study of in-situ protein folding dynamics (Lakowicz J R, Gryczynski I, Piszczek G, et al., “Microsecond dynamics of biological macromolecules,” Methods Enzym. 323, 473-509 (2000); Schauerte J A, Steel D G, Gafni A, “Time-resolved room temperature tryptophan phosphorescence in proteins,” Methods Enzym., 278, 49-71 (1997); Subramaniam V, Gafni A, Steel D G, “Time-resolved tryptophan phosphorescence spectroscopy: A sensitive probe of protein folding and structure,” IEEE J of Selected Topics in Quantum Electronics 2, 1107-1114 (1996)).
Time resolved measurement has been segmented into two areas, represented by the two modalities of imaging, FLIM and TRF: (a) Systems that measure in the ns to μs time regime. When applied to imaging, these are called fluorescent lifetime imager (FLIM) systems, where in the output image the value of each pixel represents the mean lifetime of the sample emission from that location. That is, images are ‘lifetime’ images, and are not intensity based; (b) Systems that measure in the μs to ms time regime, usually used in conjunction with bioassay TRF reagents (above refs.). No commercial cell imaging system is known in this area.
The ns time domain instrumentation for time resolved imaging has been developed for use with probes such as FITC, Rhodamine, EGFP. The systems operate by two approaches: fast pulsed laser excitation of the sample followed by fast detection (e.g., camera/image intensifier combinations), or more commonly, fast electronic modulation of a steady excitation source (e.g., laser and or diodes) in conjunction with a fast detector employing phase shift electronics (van Munster E B, Gadella T W J, “Fluorescence lifetime imaging microscopy (FLIM),” Adv in Biochem Eng/Biotech, 95, 143-175 (2005); Suhling K, French P M W, Phillips D, “Time-resolved fluorescence microscopy,” Photochem & Photobio Sci, 4 (1) 13-22 (2005); Elson D et al., “Time-domain fluorescence lifetime imaging applied to biological tissue,” Photochem & Photobio Sci, 3 (8) 795-801 (2004); Krishnan R V et al., “Development of multiphoton fluorescence lifetime imaging microscopy (FLIM) system using a streak camera,” Rev. Sci. Instrum., 74, 2714-2721 (2003); Clegg R M, “Fluorescence lifetime-resolved imaging: Measuring lifetimes in an image,” Methods in Enzymology, 360, 509-542 (2003)). Examples of prior art disclosures include WO2000008443(A1) by P Bastiaens et al. employing modulated excitation and emission constructs, commercialized through Lambert Instruments, Leutingewolde, the Netherlands (http://www.lambert-instruments.com/), and the Hamamatsu C9136 lifetime imaging microscopy system (http://sales.hamamatsu.com/), employing ps pulsed lasers and fast streak cameras, disclosed in Krishnan R V et al., “Development of multiphoton fluorescence lifetime imaging microscopy (FLIM) system using a streak camera,” Rev. Sci. Instrum., 74, 2714-2721 (2003). These systems can create high-content cellular lifetime images but are all expensive, complex, and difficult to operate and maintain.
The μs to ms time domain instrumentation systems for time resolved measurement have been developed for use with the long-lived TRF reagents (above refs.), and are known as TRF readers (or TRF imagers). The readers rely on detection with photomultiplier tubes (PMT). They have slow throughputs because they read microtiter plates, one well at a time. Another class of systems relies on macro-imaging of microtiter plates, as exemplified by the LEADSEEKER™ Multimodality Imaging System (GE® Healthcare Bio-Sciences, Piscataway, N.J., disclosed in US patent publication no. 2003-0160151) and the VIEWLUX™ ultra HTS Microplate Imager (PerkinElmer Life And Analytical Sciences, Inc., Wellesley, Mass.). The macro-imagers use a charge-coupled device (CCD) to capture the images. They have higher throughput than the PMT-based readers because all wells of a microtiter plate are imaged at once. Most TRF readers (or imagers) operate by using a μs flash lamp to excite the sample, along with electronic gating of the detector to read the sample fluorescence after a delay time of few us, for a gate duration of about 1-3 emission lifetime. In some systems the flash lamp is replaced by the mechanical chopping of a steady light source and the emission itself. CCDs are relatively slow-reading devices so that the gating of TRF imagers is accomplished outside the detector, either by an optoelectronic shutter (as in the LEADSEEKER™), or by a mechanical chopper (as in the VIEWLUX™)
The key advantage of TRF imagers is in reduction of the contribution of background light to overall signal intensity. The images created are intensity images and the gated signal is dependent on the instrumental settings. TRF imagers are usually not used as FLIM lifetime measuring systems. However, creation of a lifetime image from TRF imagers is possible in principle. It requires acquisition of multiple images with different gating times (or delay times), and further mathematical processing of the image data for each pixel position to extract a mean lifetime value for that position.
Integration of the TRF technology into microscopy for high content imaging has been disclosed in U.S. Pat. No. 5,523,573, and Seveus L et al., “Time-resolved fluorescence imaging of europium chelate label in immunohistochemistry and in situ hybridization,” Cytometry, 13, 329-338 (1992); Soini A E et al., “A new technique for multiparametric imaging microscopy: Use of long decay time photoluminescent labels enables multiple color immunocytochemistry with low channel to-channel crosstalk,” Microsc Res Technol, 62, 396-407 (2003). In this approach, the excitation light is pulsed by use of, either a laser or flash lamp, or a revolving shutter in front of a steady lamp, and detection is gated by use of timing electronics and a mechanical chopper in front of the emission light. These components add cost and complexity to the base microscopic imaging system. Moreover, the uses of a rotating chopper limits the detection system to the ms time domain and longer, while introducing safety concerns and the possibility of image distortion from mechanical vibrations. For these reasons, at present no commercial high-content TRF microscopic imagers are offered on the market.
Therefore, there is a need for a system that overcomes the expense and complexities of FLIM systems and the limitations of TRF imagers by devising a system that adds time resolution capability with little additional cost to the base steady-state fluorescence imaging system.